Methods for determining carrier status

ABSTRACT

The invention generally relates to methods for determining carrier status with respect to a condition or disease. In certain embodiments, the method involves exposing a sample to a plurality of molecular inversion probes capable of capturing DNA from at least one genomic region suspected of having an altered copy number and at least one internal control DNA known or suspected to have a stable copy number, capturing and sequencing DNA that binds to the molecular inversion probes, and determining a copy number state of the at least one genomic region based on the sequence results.

FIELD

The present invention relates generally to genetic testing, and, moreparticularly, to methods for determining carrier or disease status withrespect to a particular disease or condition.

BACKGROUND

Genetic counseling is the process by which individuals, typicallyprospective parents, are advised regarding the likelihood oftransmitting an inherited or genetic disorder to any future offspring.To assist in the process, individuals seeking counseling typicallyundergo a number of tests that assess indications of genetic disorders.These tests may include, for example, screening assays that detectclinically significant variations in various biomarkers. For example,carrier screening can determine if members of a couple are both carriersof a recessive genetic disorder. With this information, the couple canlearn or rule out that they are at risk for having children with thegenetic disorder.

Some currently available tests screen for autosomal recessive disorders,including spinal muscular atrophy (SMA). Spinal muscular atrophy (SMA)is an autosomal recessive disease characterized by degeneration of motorneurons in the anterior horn of a spinal cord, leading to muscularparalysis and atrophy. The prognosis for SMA may vary on an individualbasis. However, the majority of patients children diagnosed with SMA donot reach the age of 10. SMA is the second most common autosomalrecessive inherited disorder in humans and the most common genetic causeof infant mortality.

SMA is caused by the homozygous deletion or mutations of the survivalmotor neuron gene (SMN), including telomeric SMN (SMN1) and centromericSMN (SMN2) genes. SMA has a carrier frequency of approximately 1 in 40,which is attributable primarily to SMN1 copy number loss produced byeither deletion of part or all of SMN1, or conversion of SMN1 to theSMN2 gene, a linked paralog that encodes an identical protein but ispoorly expressed due to a silent coding variant that disrupts propersplicing.

Current methods for clinical SMA carrier screening include multiplexligation-dependent probe amplification (MLPA) for assessing the copynumber state of SMN1 in a manner that distinguishes between SMN1 andSMN2, typically by interrogating the exon 7 variant that differs betweenthe paralogs. MLPA is a variation of multiplex polymerase chain reaction(PCR) and permits multiple targets to be amplified with only a singleprimer pair.

Although current MLPA-based carrier screening assays may be relativelysensitive and specific, they have significant drawbacks. For example,one problem with current MLPA-based assays is that they generallyquantify only the nucleotide difference in exon 7 of the SMN1/SMN2genes. However, it has been found that SMA in approximately 6% ofaffected patients is caused by point mutations at other exons in whichSMN1 is present. As such, MLPA-based assays may miss detection of suchpoint mutations, possibly resulting in inaccurate screening results.Another problem is that MLPA-based assays are time consuming andrelatively low-throughput.

SUMMARY

The invention solves problems associated with current carrier screeningassays by providing molecular inversion probes for capturing at leastone genomic region known or suspected to be associated with a diseaseand subsequently sequencing the captured DNA to determine the copynumber state of the captured DNA. In certain aspects, the captured DNAmay be sequenced by known high-throughput sequence methods ortechniques. The carrier status for the disease can then be determinedbased on the identified copy number state. In particular, the copynumber state may be indicative of whether the patient is a carrier of aparticular disease or condition for which copy number variation isdiagnostic, such as an autosomal recessive trait (e.g., spinal muscularatrophy). For example, it can be determined whether copy numbervariation exists in the genomic regions of interest based on theidentified copy number, thereby providing a method for determiningwhether the patient is a carrier of such an autosomal recessive trait.

Accordingly, the invention overcomes the problems of current carrierscreening assays, particularly MLPA-based assays, based, at least inpart, on compatibility with automated high-throughput screening. Inparticular, the invention provides the sensitivity and specificity fordetection of copy number variation in SMN1 and/or SMN2, similar toMLPA-based methods, by utilizing molecular inversion probes.Additionally, by using high-throughput screening methods, the inventionallows detection of deleterious SMN1 point mutations and indels thatwould otherwise be missed by MLPA and related approaches, therebyproviding more reliable and accurate diagnosis.

In certain aspects, the invention provides a method for determining copynumber state of one or more genes in a sample. The method includesexposing a sample to a plurality of molecular inversion probes capableof capturing DNA from at least one genomic region suspected of having analtered copy number, and at least one internal control DNA known orsuspected to have a stable copy number. The at least one genomic regionmay be associated with a particular locus, such as, for example, SMN1 orSMN2, that is associated with a condition or disease.

In embodiments that utilize molecular inversion probes, any molecularinversion probe may be used. An exemplary MIP is a single-stranded probeabout 70 nucleotides in length, composed of a universal core of 30nucleotides that is flanked by specific 20-nucleotide targetingsequences on each side, i.e. targeting arms. However, the length andcomposition of the probe can vary to most adequately capture the desiredtarget sequence. The targeting arms are designed to hybridize tospecific genomic regions upstream and downstream of a target sequence ofinterest located on the nucleic acid fragment. After the target sequenceof interest is isolated between the target arms, the target sequence canbe analyzed. Although each MIP captures one target of interest foranalysis, multiple probes can be combined into a single vesselcontaining sample for a multiplexed assay that simultaneously examinesmultiple target loci.

Upon capturing DNA that binds the molecular inversion probes, thecaptured DNA is sequenced and further analyzed to determine copy numberstate for use in determining carrier status of a disease in which copynumber variation is diagnostic. The sequencing results in a plurality ofreads for both the genomic region suspected of having an altered copynumber and the internal control DNA.

Read counts obtained in the sequencing step are normalized for thegenomic region with respect to the internal control DNA. The normalizedread counts for the genomic region and the control DNA are compared withone another to obtain a ratio of normalized read count of genomicregions of interest/normalized read count of internal control DNA. Thecopy number state of the genomic region can then be determined basedupon the ratio (i.e. difference between the normalized read counts).

The copy number state can be used to determine the carrier status of anindividual from which the sample was obtained. In particular, the copynumber state may be indicative of whether the patient is a carrier of aparticular disease or condition for which copy number variation isdiagnostic. Copy-number variations (CNVs), a form of structuralvariation, are alterations of the DNA of a genome that results in thecell having an abnormal number of copies of one or more sections of theDNA. CNVs correspond to relatively large regions of the genome that havebeen deleted (fewer than the normal number) or duplicated (more than thenormal number) on certain chromosomes. Some diseases are associated withCNVs of particular genes or gene fragments. For example, copy numbervariation in the SMN1 and/or SMN2 gene is closely associated with spinalmuscular atrophy (SMA), an autosomal recessive trait. Accordingly,variation in the copy number state may indicate the presence of anautosomal recessive trait, thereby determining that the patient is acarrier of such an autosomal recessive trait.

In certain aspect, it can be determined whether copy number variationexists in the genomic regions of interest based on the calculated ratioof normalized read count of genomic regions of interest/normalized readcount of internal control DNA. For example, copy number variationgenerally exists if there is a statistical difference between thenormalized read counts for the genomic region the control DNA (i.e. aratio outside of a range of 0.8 to 1.2). Similarly, copy numbervariation does not exist if there is little or no statistical differencebetween the normalized read counts for the genomic region the controlDNA (i.e. a ratio within the range of 0.8 to 1.2).

The invention may be used to determine carrier status for otherautosomal recessive traits, including, but not limited to, cysticfibrosis, sickle cell anemia, tay sachs disease, Familialhyperinsulinism, Canavan Disease, Maple Syrup Urine Disease, Bloom'sSyndrome, Usher Syndrome type IIIA, Dihydrolipoamide dehydrogenasedeficiency, Fanconi anemia group C, Familial dysautonomia, MucolipidosisType IV, Usher Syndrome Type IV, Nieman-Pick disease type A/B, WalkerWarburg syndrome, and Joubert Syndrome.

In a related aspect, the invention provides a method of determining copynumber between two paralogous genes. The method includes exposing asample including a first genetic locus, a second genetic locus, and atleast one control genetic locus to a plurality of molecular inversionprobes. At least some of the probes are capable of hybridizing with oneor more of the first genetic locus, the second genetic locus, and thecontrol genetic locus. The first and second genetic locus may beassociated with a particular disease for which copy number variation isdiagnostic. Upon obtaining DNA that is hybridized to a member of theplurality of molecular inversion probes, the obtained DNA is sequenced(e.g., next-generation sequencing method) and read counts for each ofthe first genetic locus and the second genetic locus are normalized withrespect to the at least one control genetic locus. The normalized readcounts for each of the first genetic locus and the second genetic locusare then compared to normalized read counts for one or more controlsamples with a known number of copies of the first genetic locus and thesecond genetic locus. Based on the comparison, relative copy numbers ofthe first genetic locus and the second genetic locus can be determined.The determined copy numbers can then be used to determine the carrierstatus of an individual from which the sample was obtained (i.e. whetherthe patient is a carrier of the disease).

In another related aspect, the invention provides a method for thedetermination of carrier status with respect to one or more conditions.The method includes exposing a sample to a plurality of molecularinversion probes, at least some of which are capable of hybridizing toDNA of a first locus or DNA of a second locus. DNA in the sample that ishybridized to a member of the plurality of probes is obtained and thensequenced (e.g. next-generation sequencing methods) to obtain sequenceread counts for the first locus and the second locus. The read countsare normalized with respect to a control locus, the copy number of whichis stable or known. The copy number status of the first locus and thesecond locus is then inferred based, at least in part, on a comparisonof the normalized read counts for the first locus and the second locuswith a standard.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of methods of the invention.

FIG. 2 illustrates a system for performing methods of the invention.

DETAILED DESCRIPTION

The invention generally relates to methods for determining carrierstatus with respect to a condition or disease, particularly an autosomalrecessive trait. In certain embodiments, methods of the inventioninclude determining copy number state of one or more genes in a sample.The methods includes exposing a sample to a plurality of molecularinversion probes capable of capturing DNA from at least one genomicregion suspected of having an altered copy number, and at least oneinternal control DNA known or suspected to have a stable copy number. Insome embodiments, the at least one genomic region may be associated witha particular locus, such as, for example, SMN1 or SMN2, that isassociated with a condition or disease. The methods further includecapturing and sequencing DNA that binds to the molecular inversionprobes, normalizing read counts obtained in the sequencing step for theleast one genomic region with respect to the internal control DNA, anddetermining a copy number state of the at least one genomic region basedon a comparison between the normalized read counts of the genomic regionand read counts for a control diploid locus.

By way of overview, methods of the invention involve obtaining one ormore samples including nucleic acids. The nucleic acids, includinggenomic nucleic acids, of each sample are fragmented and/or denatured soas to render the nucleic acid single stranded for hybridization to acapture probe, such as a molecular inversion probe. As described ingreater detail herein, each sample is exposed to a plurality ofmolecular inversion probes to hybridize or bind with at least onegenomic region of interest (e.g. locus) located on the nucleic acidfragments. After capture of the genomic region of interest, the capturedregion is subjected to an enzymatic gap-filling and ligation step, andfurther subjected to amplification based on sample-specific barcodepolymerase chain reaction (PCR). The resulting barcodes PCRs for eachsample are then combined into a master pool and quantified. Analysis ofthe captured regions of interest involves sequencing, e.g., with anext-generation sequencer, and determining copy number states of thegenomic regions of interest based on the sequencing readout.

Nucleic acids suitable for use in aspects of the invention include butare not limited to genomic DNA, genomic RNA, synthesized nucleic acids,whole or partial genome amplification product, and high molecular weightnucleic acids, e.g. individual chromosomes. Genomic DNA and genomic RNAconstitute the total genetic information of an organism. Genomic nucleicacids molecules are generally large, and in most organisms are organizedinto DNA—protein complexes called chromosomes, which the exception ofviruses that have RNA genomes. Genomic RNA also includes, for example,RNA transcribed from DNA, unprocessed transcripts, mRNAs, and cDNAs.Sometimes the quality and quantity of genomic nucleic acids obtainedfrom samples precludes their usefulness in large scale genotypingstudies. To overcome this problem, use of whole genome amplificationproducts and partial genome amplification products allows forcharacterization of the genome of a sample even if the quantity andquality of the genomic nucleic acid is limited.

Samples and Obtaining Nucleic Acid

In certain aspects, methods of the invention may involve obtaining asample. The sample is typically a tissue or body fluid that is obtainedin any clinically acceptable manner. A tissue is a mass of connectedcells and/or extracellular matrix material, e.g. skin tissue,endometrial tissue, nasal passage tissue, CNS tissue, neural tissue, eyetissue, liver tissue, kidney tissue, placental tissue, mammary glandtissue, placental tissue, gastrointestinal tissue, musculoskeletaltissue, genitourinary tissue, bone marrow, and the like, derived from,for example, a human or other mammal and includes the connectingmaterial and the liquid material in association with the cells and/ortissues. A body fluid is a liquid material derived from, for example, ahuman or other mammal. Such body fluids include, but are not limited to,mucous, blood, plasma, serum, serum derivatives, bile, blood, maternalblood, phlegm, saliva, sweat, amniotic fluid, menstrual fluid, mammaryfluid, follicular fluid of the ovary, fallopian tube fluid, peritonealfluid, urine, and cerebrospinal fluid (CSF), such as lumbar orventricular CSF. A sample may also be a fine needle aspirate or biopsiedtissue. A sample also may be media containing cells or biologicalmaterial. A sample may also be a blood clot, for example, a blood clotthat has been obtained from whole blood after the serum has beenremoved. Samples are also obtained from the environment (e.g., air,agricultural, water and soil); and research samples (e.g., products of anucleic acid amplification reaction, or purified genomic DNA, RNA,proteins, etc.).

Isolation, extraction or derivation of genomic nucleic acids isperformed by methods known in the art. Isolating nucleic acid from abiological sample generally includes treating a biological sample insuch a manner that genomic nucleic acids present in the sample areextracted and made available for analysis. Any isolation method thatresults in extracted/isolated genomic nucleic may be used in thepractice of the present invention.

Nucleic acids may be obtained by methods known in the art. Generally,nucleic acids are extracted using techniques, such as those described inSambrook, J. Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning:A Laboratory Manual. 2nd ed. Cold Spring Harbor, N.Y.: Cold SpringHarbor Laboratory.), the contents of which are incorporated by referenceherein. Other methods include: salting out DNA extraction (P. Sunnuckset al., Genetics, 1996, 144: 747-756; S. M. Aljanabi and I. Martinez,Nucl. Acids Res. 1997, 25: 4692-4693), trimethylammonium bromide saltsDNA extraction (S. Gustincich et al., BioTechniques, 1991, 11: 298-302)and guanidinium thiocyanate DNA extraction (J. B. W. Hammond et al.,Biochemistry, 1996, 240: 298-300). Several protocols have been developedto extract genomic DNA from blood.

There are also numerous kits that can be used to extract DNA fromtissues and bodily fluids and that are commercially available from, forexample, BD Biosciences Clontech (Palo Alto, Calif.), EpicentreTechnologies (Madison, Wis.), Gentra Systems, Inc. (Minneapolis, Minn.),MicroProbe Corp. (Bothell, Wash.), Organon Teknika (Durham, N.C.),Qiagen Inc. (Valencia, Calif.), Autogen (Holliston, Mass.); BeckmanCoulter (Brea, Calif.), (AutoGenFlex STAR robot with Qiagen FlexiGenechemistry. For example, Autogen manufactures FlexStar automatedextraction kits used in combination with Qiagen FlexiGene Chemistry, andBeckeman Coulter manufactures Agencourt GenFind kits for bead-basedextraction chemistry. User Guides that describe in detail theprotocol(s) to be followed are usually included in all these kits, forexample, Qiagen's literature for their PureGene extraction chemistryentitled “Qiagen PureGene Handbook” 3rd Edition, dated June 2011.

After cells have been obtained from the sample, it is preferable to lysecells in order to isolate genomic nucleic acid. Cellular extracts can besubjected to other steps to drive nucleic acid isolation towardcompletion by, e.g., differential precipitation, column chromatography,extraction with organic solvents and the like. Extracts then may befurther treated, for example, by filtration and/or centrifugation and/orwith chaotropic salts such as guanidinium isothiocyanate or urea or withorganic solvents such as phenol and/or HCCl₃ to denature anycontaminating and potentially interfering proteins. The genomic nucleicacid can also be resuspended in a hydrating solution, such as an aqueousbuffer. The genomic nucleic acid can be suspended in, for example,water, Tris buffers, or other buffers. In certain embodiments thegenomic nucleic acid can be re-suspended in Qiagen DNA hydrationsolution, or other Tris-based buffer of a pH of around 7.5.

Depending on the type of method used for extraction, the genomic nucleicacid obtained can vary in size. The integrity and size of genomicnucleic acid can be determined by pulse-field gel electrophoresis (PFGE)using an agarose gel.

In addition to genomic nucleic acids, whole genome amplification productand partial genomic amplification products can be used in aspects of theinvention. Methods of obtaining whole genome amplification product andpartial genome amplification product are described in detail in Pinteret al. U.S. Patent Publication Number 2004/0209299, and include, forexample, generally ligation mediated PCR™, random primed PCR™, stranddisplacement mediated PCR™, and cell immortalization.

In certain embodiments, a genomic sample is collected from a subjectfollowed by enrichment for genes or gene fragments of interest, forexample by hybridization to a nucleotide array. The sample may beenriched for genes of interest using methods known in the art, such ashybrid capture. See for examples, Lapidus (U.S. Pat. No. 7,666,593), thecontent of which is incorporated by reference herein in its entirety. Aswill be described in more detail below, a preferable capture method usesmolecular inversion probes.

Fragmenting the Nucleic Acid

Nucleic acids, including genomic nucleic acids, can be fragmented usingany of a variety of methods, such as mechanical fragmenting, chemicalfragmenting, and enzymatic fragmenting. Methods of nucleic acidfragmentation are known in the art and include, but are not limited to,DNase digestion, sonication, mechanical shearing, and the like (J.Sambrook et al., “Molecular Cloning: A Laboratory Manual”, 1989,2.sup.nd Ed., Cold Spring Harbour Laboratory Press: New York, N.Y.; P.Tijssen, “Hybridization with Nucleic Acid Probes—Laboratory Techniquesin Biochemistry and Molecular Biology (Parts I and II)”, 1993, Elsevier;C. P. Ordahl et al., Nucleic Acids Res., 1976, 3: 2985-2999; P. J.Oefner et al., Nucleic Acids Res., 1996, 24: 3879-3889; Y. R.Thorstenson et al., Genome Res., 1998, 8: 848-855). U.S. PatentPublication 2005/0112590 provides a general overview of various methodsof fragmenting known in the art.

Genomic nucleic acids can be fragmented into uniform fragments orrandomly fragmented. In certain aspects, nucleic acids are fragmented toform fragments having a fragment length of about 5 kilobases or 100kilobases. In one embodiment, the genomic nucleic acid fragments canrange from 1 kilobases to 20 kilobases. Fragments can vary in size andhave an average fragment length of about 10 kilobases. However, desiredfragment length and ranges of fragment lengths can be adjusted dependingon the type of nucleic acid targets one seeks to capture and the designand type of MIP probes. The particular method of fragmenting is selectedto achieve the desired fragment length. Numerous non-limiting examplesare provided below.

Chemical fragmentation of genomic nucleic acids can be achieved using anumber of different methods. For example, hydrolysis reactions includingbase and acid hydrolysis are common techniques used to fragment nucleicacid. Hydrolysis is facilitated by temperature increases, depending uponthe desired extent of hydrolysis. Fragmentation can be accomplished byaltering temperature and pH as described below. The benefit of pH-basedhydrolysis for shearing is that it can result in single-strandedproducts. Additionally, temperature can be used with certain buffersystems (e.g. Tris) to temporarily shift the pH up or down from neutralto accomplish the hydrolysis, then back to neutral for long-term storageetc. Both pH and temperature can be modulated to effect differingamounts of shearing (and therefore varying length distributions).

In one aspect, a nucleic acid is fragmented by heating a nucleic acidimmersed in a buffer system at a certain temperature for a certainperiod to time to initiate hydrolysis and thus fragment the nucleicacid. The pH of the buffer system, duration of heating, and temperaturecan be varied to achieve a desired fragmentation of the nucleic acid. Inone embodiment, after a genomic nucleic acid is purified, it isresuspended in a Tris-based buffer at a pH between 7.5 and 8.0, such asQiagen's DNA hydrating solution. The resuspended genomic nucleic acid isthen heated to 65° C. and incubated overnight (about 16-24 hours) at 65°C. Heating shifts the pH of the buffer into the low- to mid-6 range,which leads to acid hydrolysis. Over time, the acid hydrolysis causesthe genomic nucleic acid to fragment into single-stranded and/ordouble-stranded products.

Other methods of hydrolytic fragmenting of nucleic acids includealkaline hydrolysis, formalin fixation, hydrolysis by metal complexes(e.g., porphyrins), and/or hydrolysis by hydroxyl radicals. RNA shearsunder alkaline conditions, see, e.g. Nordhoff et al., Nucl. Acid. Res.,21 (15):3347-57 (2003), whereas DNA can be sheared in the presence ofstrong acids or strong bases.

An exemplary acid/base hydrolysis protocol for producing genomic nucleicacid fragments is described in Sargent et al. (1988) Methods Enzymol.,152:432. Briefly, 1 g of purified DNA is dissolved in 50 mL 0.1 N NaOH.1.5 mL concentrated HCl is added, and the solution is mixed quickly. DNAwill precipitate immediately, and should not be stirred for more than afew seconds to prevent formation of a large aggregate. The sample isincubated at room temperature for 20 minutes to partially depurinate theDNA. Subsequently, 2 mL 10 N NaOH ([OH—] concentration to 0.1 N) isadded, and the sample is stirred until the DNA redis solves completely.The sample is then incubated at 65° C. for 30 minutes in order tohydrolyze the DNA. Resulting fragments typically range from about250-1000 nucleotides but can vary lower or higher depending on theconditions of hydrolysis.

Chemical cleavage can also be specific. For example, selected nucleicacid molecules can be cleaved via alkylation, particularlyphosphorothioate-modified nucleic acid molecules (see, e.g., K. A.Browne, “Metal ion-catalyzed nucleic Acid alkylation and fragmentation,”J. Am. Chem. Soc. 124(27):7950-7962 (2002)). Alkylation at thephosphorothioate modification renders the nucleic acid moleculesusceptible to cleavage at the modification site. See I. G. Gut and S.Beck, “A procedure for selective DNA alkylation and detection by massspectrometry,” Nucl. Acids Res. 23(8):1367-1373 (1995).

Methods of the invention also contemplate chemically shearing nucleicacids using the technique disclosed in Maxam-Gilbert Sequencing Method(Chemical or Cleavage Method), Proc. Natl. Acad. Sci. USA. 74:560-564.In that protocol, the genomic nucleic acid can be chemically cleaved byexposure to chemicals designed to fragment the nucleic acid at specificbases, such as preferential cleaving at guanine, at adenine, at cytosineand thymine, and at cytosine alone.

Mechanical shearing of nucleic acids into fragments can occur using anymethod known in the art. For example, fragmenting nucleic acids can beaccomplished by hydroshearing, trituration through a needle, andsonication. See, for example, Quail, et al. (November 2010) DNA:Mechanical Breakage. In: eLS. John Wiley & Sons, Chichester.doi:10.1002/9780470015902.a0005 333.pub2.

The nucleic acid can also be sheared via nebulization, see (Roe, B A,Crabtree. J S and Khan, A S 1996); Sambrook & Russell, Cold Spring HarbProtoc 2006. Nebulizing involves collecting fragmented DNA from a mistcreated by forcing a nucleic acid solution through a small hole in anebulizer. The size of the fragments obtained by nebulization isdetermined chiefly by the speed at which the DNA solution passes throughthe hole, altering the pressure of the gas blowing through thenebulizer, the viscosity of the solution, and the temperature. Theresulting DNA fragments are distributed over a narrow range of sizes(700-1330 bp). Shearing of nucleic acids can be accomplished by passingobtained nucleic acids through the narrow capillary or orifice (Oefneret al., Nucleic Acids Res. 1996; Thorstenson et al., Genome Res. 1995).This technique is based on point—sink hydrodynamics that result when anucleic acid sample is forced through a small hole by a syringe pump.

In HydroShearing (Genomic Solutions, Ann Arbor, Mich., USA), DNA insolution is passed through a tube with an abrupt contraction. As itapproaches the contraction, the fluid accelerates to maintain thevolumetric flow rate through the smaller area of the contraction. Duringthis acceleration, drag forces stretch the DNA until it snaps. The DNAfragments until the pieces are too short for the shearing forces tobreak the chemical bonds. The flow rate of the fluid and the size of thecontraction determine the final DNA fragment sizes.

Sonication is also used to fragment nucleic acids by subjecting thenucleic acid to brief periods of sonication, i.e. ultrasound energy. Amethod of shearing nucleic acids into fragments by sonification isdescribed in U.S. Patent Publication 2009/0233814. In the method, apurified nucleic acid is obtained placed in a suspension havingparticles disposed within. The suspension of the sample and theparticles are then sonicated into nucleic acid fragments.

An acoustic-based system that can be used to fragment DNA is describedin U.S. Pat. Nos. 6,719,449, and 6,948,843 manufactured by Covaris Inc.U.S. Pat. No. 6,235,501 describes a mechanical focusing acousticsonication method of producing high molecular weight DNA fragments byapplication of rapidly oscillating reciprocal mechanical energy in thepresence of a liquid medium in a closed container, which may be used tomechanically fragment the DNA.

Another method of shearing nucleic acids into fragments uses ultrasoundenergy to produce gaseous cavitation in liquids, such as shearing withDiagonnode's BioRuptor®. Cavitation is the formation of small bubbles ofdissolved gases or vapors due to the alteration of pressure in liquids.These bubbles are capable of resonance vibration and produce vigorouseddying or microstreaming. The resulting mechanical stress can lead toshearing the nucleic acid in to fragments.

Enzymatic fragmenting, also known as enzymatic cleavage, cuts nucleicacids into fragments using enzymes, such as endonucleases, exonucleases,ribozymes, and DNAzymes. Such enzymes are widely known and are availablecommercially, see Sambrook, J. Molecular Cloning: A Laboratory Manual,3rd (2001) and Roberts R J (January 1980). “Restriction and modificationenzymes and their recognition sequences,” Nucleic Acids Res. 8 (1):r63-r80. Varying enzymatic fragmenting techniques are well-known in theart, and such techniques are frequently used to fragment a nucleic acidfor sequencing, for example, Alazard et al, 2002; Bentzley et al, 1998;Bentzley et al, 1996; Faulstich et al, 1997; Glover et al, 1995;Kirpekar et al, 1994; Owens et al, 1998; Pieles et al, 1993; Schuette etal, 1995; Smirnov et al, 1996; Wu & Aboleneen, 2001; Wu et al, 1998a.

The most common enzymes used to fragment nucleic acids areendonucleases. The endonucleases can be specific for either adouble-stranded or a single stranded nucleic acid molecule. The cleavageof the nucleic acid molecule can occur randomly within the nucleic acidmolecule or can cleave at specific sequences of the nucleic acidmolecule. Specific fragmentation of the nucleic acid molecule can beaccomplished using one or more enzymes in sequential reactions orcontemporaneously.

Restriction endonucleases recognize specific sequences withindouble-stranded nucleic acids and generally cleave both strands eitherwithin or close to the recognition site in order to fragment the nucleicacid. Naturally occurring restriction endonucleases are categorized intofour groups (Types I, II III, and IV) based on their composition andenzyme cofactor requirements, the nature of their target sequence, andthe position of their DNA cleavage site relative to the target sequence.Bickle T A, Kruger D H (June 1993). “Biology of DNA restriction”.Microbiol. Rev. 57 (2): 434-50; Boyer H W (1971). “DNA restriction andmodification mechanisms in bacteria”. Annu. Rev. Microbiol. 25: 153-76;Yuan R (1981). “Structure and mechanism of multifunctional restrictionendonucleases”. Annu. Rev. Biochem. 50: 285-319. All types of enzymesrecognize specific short DNA sequences and carry out the endonucleolyticcleavage of DNA to give specific fragments with terminal 5′-phosphates.The enzymes differ in their recognition sequence, subunit composition,cleavage position, and cofactor requirements. Williams R J (2003).“Restriction endonucleases: classification, properties, andapplications”. Mol. Biotechnol. 23 (3): 225-43.

Where restriction endonucleases recognize specific sequencings indouble-stranded nucleic acids and generally cleave both strands, nickingendonucleases are capable of cleaving only one of the strands of thenucleic acid into a fragment. Nicking enzymes used to fragment nucleicacids can be naturally occurring or genetically engineered fromrestriction enzymes. See Chan et al., Nucl. Acids Res. (2011) 39 (1):1-18.

Denaturing the Nucleic Acids

Methods of the invention also provide for denaturing nucleic acid torender the nucleic acid single stranded for hybridization to a captureprobe, such as a MIP probe. Denaturation can result from thefragmentation method chosen, as described above. For example, oneskilled in the art recognizes that a genomic nucleic acid can bedenatured during pH-based shearing or fragmenting via nickingendonucleases. Denaturation can occur either before, during, or afterfragmentation. In addition, the use of pH or heat during the fragmentingstep can result in denatured nucleic acid fragments. See, for example,McDonnell, “Antisepsis, disinfection, and sterilization: types, action,and resistance,” pg. 239 (2007).

Heat-based denaturing is the process by which double-strandeddeoxyribonucleic acid unwinds and separates into single-stranded strandsthrough the breaking of hydrogen bonding between the bases. Heatdenaturation of a nucleic acid of an unknown sequence typically uses atemperature high enough to ensure denaturation of even nucleic acidshaving a very high GC content, e.g., 95° C.-98° C. in the absence of anychemical denaturant. It is well within the abilities of one of ordinaryskill in the art to optimize the conditions (e.g., time, temperature,etc.) for denaturation of the nucleic acid. Temperatures significantlylower than 95° C. can also be used if the DNA contains nicks (andtherefore sticky overhangs of low Tm) or sequence of sufficiently lowTm.

Denaturing nucleic acids with the use of pH is also well known in theart, and such denaturation can be accomplished using any method known inthe art such as introducing a nucleic acid to high or low pH, low ionicstrength, and/or heat, which disrupts base-pairing causing adouble-stranded helix to dissociate into single strands. For methods ofpH-based denaturation see, for example, Dore et al. Biophys J. 1969November; 9(11): 1281-1311; A. M. Michelson The Chemistry of Nucleosidesand Nucleotides, Academic Press, London and New York (1963).

Nucleic acids can also be denatured via electro-chemical means, forexample, by applying a voltage to a nucleic acid within a solution bymeans of an electrode. Varying methods of denaturing by applying avoltage are discussed in detail in U.S. Pat. No. 6,197,508 and U.S. Pat.No. 5,993,611.

Molecular Inversion Probe Capture

Molecular inversion probe technology is used to detect or amplifyparticular nucleic acid sequences in complex mixtures. Use of molecularinversion probes has been demonstrated for detection of singlenucleotide polymorphisms (Hardenbol et al. 2005 Genome Res 15:269-75)and for preparative amplification of large sets of exons (Porreca et al.2007 Nat Methods 4:931-6, Krishnakumar et al. 2008 Proc Natl Acad SciUSA 105:9296-301). One of the main benefits of the method is in itscapacity for a high degree of multiplexing, because generally thousandsof targets may be captured in a single reaction containing thousands ofprobes.

In certain embodiments, molecular inversion probes include a universalportion flanked by two unique targeting arms. The targeting arms aredesigned to hybridize immediately upstream and downstream of a specifictarget sequence located on a genomic nucleic acid fragment. Themolecular inversion probes are introduced to nucleic acid fragments toperform capture of target sequences located on the fragments. Accordingto the invention, fragmenting aids in capture of target nucleic acid bymolecular inversion probes. As described in greater detail herein, aftercapture of the target sequence (e.g., locus) of interest, the capturedtarget may further be subjected to an enzymatic gap-filling and ligationstep, such that a copy of the target sequence is incorporated into acircle. Capture efficiency of the MIP to the target sequence on thenucleic acid fragment can be improved by lengthening the hybridizationand gap-filing incubation periods. (See, e.g., Turner E H, et al., NatMethods. 2009 Apr. 6:1-2.).

In one embodiment of the present invention, a library of molecularinversion probes is generated, wherein the probes are used in capturingDNA of genomic regions of interests (e.g., SMN1, SMN2, control DNA). Thelibrary consists of a plurality of SMA oligonucleotide probes capable ofcapturing one or more genomic regions of interest (e.g., SMN1, SMN2 andcontrol loci) within the samples to be tested.

The result of MIP capture as described above is a library of circulartarget probes, which then can be processed in a variety of ways. In oneaspect, adaptors for sequencing can be attached during commonlinker-mediated PCR, resulting in a library with non-random, fixedstarting points for sequencing. In another aspect, for preparation of ashotgun library, a common linker-mediated PCR is performed on the circletarget probes, and the post-capture amplicons are linearly concatenated,sheared, and attached to adaptors for sequencing. Methods for shearingthe linear concatenated captured targets can include any of the methodsdisclosed for fragmenting nucleic acids discussed above. In certainaspects, performing a hydrolysis reaction on the captured amplicons inthe presence of heat is the desired method of shearing for libraryproduction.

It should be appreciated that aspects of the invention can involvevarying the amounts of genomic nucleic acid and varying the amounts ofMIP probes to reach a customized result. In some embodiments, the amountof genomic nucleic acid used per subject ranges from 1 ng to 10 μg(e.g., 500 ng to 5 μg). However, higher or lower amounts (e.g., lessthan 1 ng, more than 10 μg, 10-50 μg, 50-100 μg or more) may be used. Insome embodiments, for each locus of interest, the amount of probe usedper assay may be optimized for a particular application. In someembodiments, the ratio (molar ratio, for example measured as aconcentration ratio) of probe to genome equivalent (e.g., haploid ordiploid genome equivalent, for example for each allele or for bothalleles of a nucleic acid target or locus of interest) ranges from1/100, 1/10, 1/1, 10/1, 100/1, 1000/1. However, lower, higher, orintermediate ratios may be used.

In some embodiments, the amount of target nucleic acid and probe usedfor each reaction is normalized to avoid any observed differences beingcaused by differences in concentrations or ratios. In some embodiments,in order to normalize genomic DNA and probe, the genomic DNAconcentration is read using a standard spectrophotometer or byfluorescence (e.g., using a fluorescent intercalating dye). The probeconcentration may be determined experimentally or using informationspecified by the probe manufacturer.

Similarly, once a locus has been captured, it may be amplified and/orsequenced in a reaction involving one or more primers. The amount ofprimer added for each reaction can range from 0.1 pmol to 1 nmol, 0.15pmol to 1.5 nmol (for example around 1.5 pmol). However, other amounts(e.g., lower, higher, or intermediate amounts) may be used.

In some embodiments, it should be appreciated that one or moreintervening sequences (e.g., sequence between the first and secondtargeting arms on a MIP capture probe), identifier or tag sequences, orother probe sequences that are not designed to hybridize to a targetsequence (e.g., a genomic target sequence) should be designed to avoidexcessive complementarity (to avoid cross-hybridization) to targetsequences or other sequences (e.g., other genomic sequences) that may bein a biological sample. For example, these sequences may be designed tohave a sufficient number of mismatches with any genomic sequence (e.g.,at least 5, 10, 15, or more mismatches out of 30 bases) or to have a Tm(e.g., a mismatch Tm) that is lower (e.g., at least 5, 10, 15, 20, ormore degrees C. lower) than the hybridization reaction temperature.

It should be appreciated that a targeting arm as used herein may bedesigned to hybridize (e.g., be complementary) to either strand of agenetic locus of interest if the nucleic acid being analyzed is DNA(e.g., genomic DNA). However, in the context of MIP probes, whicheverstrand is selected for one targeting arm will be used for the other one.However, in the context of RNA analysis, it should be appreciated that atargeting arm should be designed to hybridize to the transcribed RNA. Italso should be appreciated that MIP probes referred to herein as“capturing” a target sequence are actually capturing it bytemplate-based synthesis rather than by capturing the actual targetmolecule (other than for example in the initial stage when the armshybridize to it or in the sense that the target molecule can remainbound to the extended MIP product until it is denatured or otherwiseremoved).

It should be appreciated that in some embodiments a targeting arm mayinclude a sequence that is complementary to one allele or mutation(e.g., a SNP or other polymorphism, a mutation, etc.) so that the probewill preferentially hybridize (and capture) target nucleic acids havingthat allele or mutation. However, in many embodiments, each targetingarm is designed to hybridize (e.g., be complementary) to a sequence thatis not polymorphic in the subjects of a population that is beingevaluated. This allows target sequences to be captured and/or sequencedfor all alleles and then the differences between subjects (e.g., callsof heterozygous or homozygous for one or more loci) can be based on thesequence information and/or the frequency as described herein.

It should be appreciated that sequence tags (also referred to asbarcodes) may be designed to be unique in that they do not appear atother positions within a probe or a family of probes and they also donot appear within the sequences being targeted. Thus they can be used touniquely identify (e.g., by sequencing or hybridization properties)particular probes having other characteristics (e.g., for particularsubjects and/or for particular loci).

It also should be appreciated that in some embodiments probes or regionsof probes or other nucleic acids are described herein as includingcertain sequences or sequence characteristics (e.g., length, otherproperties, etc.). In addition, components (e.g., arms, central regions,tags, primer sites, etc., or any combination thereof) of such probes caninclude certain sequences or sequence characteristics that consist ofone or more characteristics (e.g., length or other properties, etc.).

As disclosed herein, uniformity and reproducibility can be increased bydesigning multiple probes per target, such that each base in the targetis captured by more than one probe. In some embodiments, the disclosureprovides multiple MIPs per target to be captured, where each MIP in aset designed for a given target nucleic acid has a central region and a5′ region and 3′ region (‘targeting arms’) which hybridize to (at leastpartially) different nucleic acids in the target nucleic acid(immediately flanking a subregion of the target nucleic acid). Thus,differences in efficiency between different targeting arms and fill-insequences may be averaged across multiple MIPs for a single target,which results in more uniform and reproducible capture efficiency.

In some embodiments, the methods involve designing a single probe foreach target (a target can be as small as a single base or as large as akilobase or more of contiguous sequence).

It may be preferable, in some cases, to design probes to capturemolecules (e.g., target nucleic acids or subregions thereof) havinglengths in the range of 1-200 bp (as used herein, a by refers to a basepair on a double-stranded nucleic acid—however, where lengths areindicated in bps, it should be appreciated that single-stranded nucleicacids having the same number of bases, as opposed to base pairs, inlength also are contemplated by the invention). However, probe design isnot so limited. For example, probes can be designed to capture targetshaving lengths in the range of up to 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 1000, or more bps, in some cases.

It is to be appreciated that the length of a capture molecule on anucleic acid fragment (e.g., a target nucleic acid or subregion thereof)is selected based upon multiple considerations. For example, whereanalysis of a target involves sequencing, e.g., with a next-generationsequencer, the target length should typically match the sequencingread-length so that shotgun library construction is not necessary.However, it should be appreciated that captured nucleic acids may besequenced using any suitable sequencing technique as aspects of theinvention are not limited in this respect.

It is also to be appreciated that some target nucleic acids on a nucleicacid fragment are too large to be captured with one probe. Consequently,it may be necessary to capture multiple subregions of a target nucleicacid in order to analyze the full target.

In some embodiments, a sub-region of a target nucleic acid is at least 1bp. In other embodiments, a subregion of a target nucleic acid is atleast 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600,700, 800, 900, 1000 bp or more. In other embodiments, a subregion of atarget nucleic acid has a length that is up to 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, or more percent of a target nucleic acidlength.

The skilled artisan will also appreciate that consideration is made, inthe design of MIPs, for the relationship between probe length and targetlength. In some embodiments, MIPs are designed such that they areseveral hundred basepairs (e.g., up to 100, 200, 300, 400, 500, 600,700, 800, 900, 1000 bp or more) longer than corresponding target (e.g.,subregion of a target nucleic acid, target nucleic acid). In someembodiments, lengths of subregions of a target nucleic acid may differ.

For example, if a target nucleic acid contains regions for which probehybridization is not possible or inefficient, it may be necessary to useprobes that capture subregions of one or more different lengths in orderto avoid hybridization with problematic nucleic acids and capturenucleic acids that encompass a complete target nucleic acid.

Methods of the invention also provide for combining the method offragmenting the nucleic acid prior to capture with other MIP capturetechniques that are designed to increase target uniformity,reproducibility, and specificity. Other MIP capture techniques are shownin co-owned and pending application, U.S. patent application Ser. No.13/266,862, “Methods and Compositions for Evaluating Genetic Markers.”

For example, multiple probes, e.g., MIPs, can be used to amplify eachtarget nucleic acid. In some embodiments, the set of probes for a giventarget can be designed to ‘tile’ across the target, capturing the targetas a series of shorter sub targets. In some embodiments, where a set ofprobes for a given target is designed to ‘tile’ across the target, someprobes in the set capture flanking non-target sequence). Alternately,the set can be designed to ‘stagger’ the exact positions of thehybridization regions flanking the target, capturing the full target(and in some cases capturing flanking non-target sequence) with multipleprobes having different targeting arms, obviating the need for tiling.The particular approach chosen will depend on the nature of the targetset. For example, if small regions are to be captured, a staggered-endapproach might be appropriate, whereas if longer regions are desired,tiling might be chosen. In all cases, the amount of bias-tolerance forprobes targeting pathological loci can be adjusted by changing thenumber of different MIPs used to capture a given molecule.

Probes for MIP capture reactions may be synthesized on programmablemicroarrays because of the large number of sequences required. Becauseof the low synthesis yields of these methods, a subsequent amplificationstep is required to produce sufficient probe for the MIP amplificationreaction. The combination of multiplex oligonucleotide synthesis andpooled amplification results in uneven synthesis error rates andrepresentational biases. By synthesizing multiple probes for eachtarget, variation from these sources may be averaged out because not allprobes for a given target will have the same error rates and biases.

Barcode PCR

With these methods, a single copy of a specific target nucleic acid maybe amplified to a level that can be sequenced. Further, the amplifiedsegments created by an amplification process such as PCR may be,themselves, efficient templates for subsequent PCR amplifications.

Amplification or sequencing adapters or barcodes, or a combinationthereof, may be attached to the fragmented nucleic acid. Such moleculesmay be commercially obtained, such as from Integrated DNA Technologies(Coralville, Iowa). In certain embodiments, such sequences are attachedto the template nucleic acid molecule with an enzyme such as a ligase.Suitable ligases include T4 DNA ligase and T4 RNA ligase, availablecommercially from New England Biolabs (Ipswich, Mass.). The ligation maybe blunt ended or via use of complementary overhanging ends. In certainembodiments, following fragmentation, the ends of the fragments may berepaired, trimmed (e.g. using an exonuclease), or filled (e.g., using apolymerase and dNTPs) to form blunt ends. In some embodiments, endrepair is performed to generate blunt end 5′ phosphorylated nucleic acidends using commercial kits, such as those available from EpicentreBiotechnologies (Madison, Wis.). Upon generating blunt ends, the endsmay be treated with a polymerase and dATP to form a template independentaddition to the 3′-end and the 5′-end of the fragments, thus producing asingle A overhanging. This single A can guide ligation of fragments witha single T overhanging from the 5′-end in a method referred to as T-Acloning. Alternatively, because the possible combination of overhangsleft by the restriction enzymes are known after a restriction digestion,the ends may be left as-is, i.e., ragged ends. In certain embodimentsdouble stranded oligonucleotides with complementary overhanging ends areused.

In certain embodiments, one or more bar code is attached to each, any,or all of the fragments. A bar code sequence generally includes certainfeatures that make the sequence useful in sequencing reactions. The barcode sequences are designed such that each sequence is correlated to aparticular portion of nucleic acid, allowing sequence reads to becorrelated back to the portion from which they came. Methods ofdesigning sets of bar code sequences is shown for example in U.S. Pat.No. 6,235,475, the contents of which are incorporated by referenceherein in their entirety. In certain embodiments, the bar code sequencesrange from about 5 nucleotides to about 15 nucleotides. In a particularembodiment, the bar code sequences range from about 4 nucleotides toabout 7 nucleotides. In certain embodiments, the bar code sequences areattached to the template nucleic acid molecule, e.g., with an enzyme.The enzyme may be a ligase or a polymerase, as discussed above.Attaching bar code sequences to nucleic acid templates is shown in U.S.Pub. 2008/0081330 and U.S. Pub. 2011/0301042, the content of each ofwhich is incorporated by reference herein in its entirety. Methods fordesigning sets of bar code sequences and other methods for attaching barcode sequences are shown in U.S. Pat. Nos. 6,138,077; 6,352,828;5,636,400; 6,172,214; 6,235,475; 7,393,665; 7,544,473; 5,846,719;5,695,934; 5,604,097; 6,150,516; RE39,793; 7,537,897; 6172,218; and5,863,722, the content of each of which is incorporated by referenceherein in its entirety. After any processing steps (e.g., obtaining,isolating, fragmenting, amplification, or barcoding), nucleic acid canbe sequenced.

Sequencing

Sequencing may be by any method known in the art. DNA sequencingtechniques include classic dideoxy sequencing reactions (Sanger method)using labeled terminators or primers and gel separation in slab orcapillary, sequencing by synthesis using reversibly terminated labelednucleotides, pyrosequencing, 454 sequencing, Illumina/Solexa sequencing,allele specific hybridization to a library of labeled oligonucleotideprobes, sequencing by synthesis using allele specific hybridization to alibrary of labeled clones that is followed by ligation, real timemonitoring of the incorporation of labeled nucleotides during apolymerization step, polony sequencing, and SOLiD sequencing. Separatedmolecules may be sequenced by sequential or single extension reactionsusing polymerases or ligases as well as by single or sequentialdifferential hybridizations with libraries of probes.

A sequencing technique that can be used includes, for example, Illuminasequencing. Illumina sequencing is based on the amplification of DNA ona solid surface using fold-back PCR and anchored primers. Genomic DNA isfragmented, and adapters are added to the 5′ and 3′ ends of thefragments. DNA fragments that are attached to the surface of flow cellchannels are extended and bridge amplified. The fragments become doublestranded, and the double stranded molecules are denatured. Multiplecycles of the solid-phase amplification followed by denaturation cancreate several million clusters of approximately 1,000 copies ofsingle-stranded DNA molecules of the same template in each channel ofthe flow cell. Primers, DNA polymerase and four fluorophore-labeled,reversibly terminating nucleotides are used to perform sequentialsequencing. After nucleotide incorporation, a laser is used to excitethe fluorophores, and an image is captured and the identity of the firstbase is recorded. The 3′ terminators and fluorophores from eachincorporated base are removed and the incorporation, detection andidentification steps are repeated. Sequencing according to thistechnology is described in U.S. Pat. No. 7,960,120; U.S. Pat. No.7,835,871; U.S. Pat. No. 7,232,656; U.S. Pat. No. 7,598,035; U.S. Pat.No. 6,911,345; U.S. Pat. No. 6,833,246; U.S. Pat. No. 6,828,100; U.S.Pat. No. 6,306,597; U.S. Pat. No. 6,210,891; U.S. Pub. 2011/0009278;U.S. Pub. 2007/0114362; U.S. Pub. 2006/0292611; and U.S. Pub.2006/0024681, each of which are incorporated by reference in theirentirety.

Sequencing generates a plurality of reads. Reads generally includesequences of nucleotide data less than about 150 bases in length, orless than about 90 bases in length. In certain embodiments, reads arebetween about 80 and about 90 bases, e.g., about 85 bases in length. Insome embodiments, these are very short reads, i.e., less than about 50or about 30 bases in length.

Data Analysis

The sequence reads are analyzed to determine copy number states ofgenomic regions of interest. A set of sequence reads can be analyzed byany suitable method known in the art. For example, in some embodiments,sequence reads are analyzed by hardware or software provided as part ofa sequence instrument. In some embodiments, individual sequence readsare reviewed by sight (e.g., on a computer monitor). A computer programmay be written that pulls an observed genotype from individual reads. Incertain embodiments, analyzing the reads includes assembling thesequence reads and then genotyping the assembled reads.

Sequence assembly can be done by methods known in the art includingreference-based assemblies, de novo assemblies, assembly by alignment,or combination methods. Assembly can include methods described in U.S.Pat. No. 8,209,130 titled Sequence Assembly by Porecca and Kennedy, thecontents of each of which are hereby incorporated by reference in theirentirety for all purposes. In some embodiments, sequence assembly usesthe low coverage sequence assembly software (LOCAS) tool described byKlein, et al., in LOCAS-A low coverage sequence assembly tool forre-sequencing projects, PLoS One 6(8) article 23455 (2011), the contentsof which are hereby incorporated by reference in their entirety.Sequence assembly is described in U.S. Pat. No. 8,165,821; U.S. Pat. No.7,809,509; U.S. Pat. No. 6,223,128; U.S. Pub. 2011/0257889; and U.S.Pub. 2009/0318310, the contents of each of which are hereby incorporatedby reference in their entirety.

As part of the analysis and determination of copy number states andsubsequent identification of copy number variation, the sequence readcounts for genomic regions of interest are normalized based on internalcontrols. In particular, an intra-sample normalization is performed tocontrol for variable sequencing depths between samples. The sequenceread counts for each genomic region of interest within a sample will benormalized according to the total read count across all controlreferences within the sample.

After normalizing read counts for both the genomic regions of interestand control references, copy number states may be determined. In oneembodiment, the normalized values for each sample of interest will becompared to the normalized values for a control sample. A ratio, forexample, may be generated based on the comparison, wherein the ratio isindicative of copy number and further determinative of any copy numbervariation. In the event that the determined copy number of a genomicregion of interest of a particular sample falls within a tolerable level(as determined by ratio between test and control samples), it can bedetermined that genomic region of interest does not present copy numbervariation and thus the patient is at low risk for being a carrier of acondition or disease associated with such. In the event that thedetermined copy number of a genomic region of interest of a particularsample falls outside of a tolerable level, it can be determined thatgenomic region of interest does present copy number variation and thusthe patient is at risk for being a carrier of a condition or diseaseassociated with such.

Example Determination of Copy Number State of SMN1

The following example shows a preferred method of practicing theinvention.

A total of approximately 28 samples were collected from a patient todetermine carrier status with respect to spinal muscular atrophy (SMA).In one embodiment, genomic DNA was extracted from whole human bloodusing a Gentra Puregene Blood Kit and following the Puregene protocolfor DNA Purification from Whole Blood (Qiagen). The protocol can bescaled (i.e. amount of solution, duration) to accommodate the desiredamount of whole genomic DNA. The samples were collected via any methodspreviously described herein. Further, it should be noted that the DNAcould be collected from other types of samples (e.g., tissue, mucous,etc.).

Of the 28 samples, there is 1 water negative control and 7 control DNAsamples and 20 test samples. Each of the control samples includes two ormore genomic regions of interest (e.g. loci) having known (or stable)copy numbers. The details of each control sample are included in Table 1below:

TABLE 1 Control Samples Control Sample ID Locus Number of Loci CopyNumber Controls 1-4 (each) Control 17 2 SMN1 5 2 SMN2 5 2 Control 5Control 17 2 SMN1 5 0 Control 6 Control 17 2 SMN1 5 1 Control 7 Control17 2 SMN1 5  3+

Control samples 1-4 each include control loci and survival motor neurongenes (SMN), including telomeric SMN (SMN1) and centromeric SMN (SMN2)genes. There are a total of 17 control loci, 5 SMN1, and 5 SMN2, all ofwhich have a known copy number of 2. Control sample 5 includes 17control loci, each having a known copy number of 2, and 5 SMN1, eachhaving a known copy number of 0. Control sample 6 includes 17 controlloci, each having a known copy number of 2, and 5 SMN1, each having aknown copy number of 1. Control sample 7 includes 17 control loci, eachhaving a known copy number of 2, and 5 SMN1, each having a known copynumber of 3 or more. As described in greater detail herein, inclusion ofthe control samples into the overall sample size allows identificationof copy number states and any copy number variation of SMN1 and/or SMN2captured from the test samples, thereby allowing subsequentdetermination of carrier status of a patient based on the copy numbervariation.

Each sample was first normalized by any know normalizing techniques. Thenormalized samples were then fragmented and/or denatured in preparationfor hybridizing with molecular inversion probes. The genomic DNA of eachsample was fragmented/denatured by any known method or techniquesufficient to fragment genomic DNA.

Once isolated, MIP capture probes were hybridized to isolated fragmentedgenomic DNA in each sample by introducing capture probe mix into eachsample well. In particular, the capture probe mix will generally includea plurality of SMA molecular inversion probes that are capable ofbinding to one or more of the genomic regions of interest (e.g., SMN1and SMN2) or the control DNA. A library of molecular inversion probeswas generated. The library may include a variety of different probeconfigurations. For example, one or more probes are capable ofhybridizing specifically to the control loci and one or more probes arecapable of hybridizing only to SMN1 or SMN2. Of those probes specific toSMN1 or SMN2, some are capable of producing sequences specific to thatparalog while some are not capable of producing paralog-specificsequences. It should be noted that some methods described herein mayutilized only one of these options. The library may also include one ormore probes capable of hybridizing nonspecifically to both SMN1 andSMN2. During the sequencing process, described in greater detail herein,the non-specificity may be resolved by reading out the sequence capturedby the non-specific probe, wherein the sequence may generally bespecific to either SMN1 or SMN2 (a variant present in the capturedsequence that is specific for one or the other paralog).

Diluted probes were introduced to the isolated fragmented genomic DNA ineach sample and the isolated whole genomic DNA was incubated in thediluted probe mix to promote hybridization. The time and temperature forincubation may be based on any known hybridization protocol, sufficientto result in hybridization of the probes to the DNA. After capture ofthe genomic region of interest (e.g., SMN1, SMN2) the captured region issubjected to an enzymatic gap-filling and ligation step, in accordancewith any known methods or techniques, including those generallydescribed herein. The captured material may further be purified.

The purified captured DNA is then amplified by any known amplificationmethods or techniques. In one embodiment, the purified captured DNA wasamplified using barcode-based PCR, in accordance with methods previouslydescribed herein. The resulting barcodes PCRs for each sample are thencombined into a master pool and quantified.

After PCR, portions of the PCR reactions for each sample were pooled andpurified, then quantified. In particular, the PCR reactions for allsamples were pooled in equal volumes into one master pool. The mastersample pool was then purified via a PCR cleanup protocol according tomanufacturer's instructions. For example, Qiagen QIAquick PCR cleanupkit (Qiagen) was used to purify the sample pool, in accordance with themanufacturer's instructions.

The purified pool was then run on a microfluidics-based platform forsizing, quantification and quality control of DNA, RNA, proteins andcells. In particular, the purified pool and control samples(pre-purification), were run on Agilent Bioanalyzer for the detectionand quantification of CF probe products and SMA probe products.

Next, the sample pool is prepared for sequencing. In a preferredembodiment, Illumina sequencing techniques were used. Prior tosequencing, the sample pool was reduced to 2 nM by diluting with 1×TE.Template DNA for cluster generation was prepared by combining 10 uL of0.1 N NaOH with 10 uL of 2 nM DNA library (sample pool) and incubatingsaid mixture at room temperature for 5 min. The mixture was then mixedwith 980 uL of HT1 buffer (Illumina), thereby reducing the denaturedlibrary to a concentration of 20 pM. This mixture was then mixed (e.g.,inversion) and pulse centrifuged. Next, 225 uL of the 20 pM library wasmixed with 775 uL of HT1 buffer to reduce the library pool to aconcentration of 4.5 pM. The library pool having a concentration of 4.5pM is used for on-board clustering in the sequencing described below.

The sequencing, and subsequent analysis, was carried out on the HiSeq2500/1500 system sold by Illumina, Inc. (San Diego, Calif.). Sequencingwas carried out with the TruSeq Rapid PE Cluster Kit and TruSeq RapidSBS 200 cycle kit (Illumina) and in accordance with manufacturer'sinstructions. In addition to the reagents and mixes included within thekits, additional reagents were prepared for genomic read sequencingprimers and reverse barcode sequencing primers.

The library pool undergoes sequencing under paired-end, dual-index runconditions. Sequencing generates a plurality of reads. Reads generallyinclude sequences of nucleotide data less than about 150 bases inlength, or less than about 90 bases in length. In certain embodiments,reads are between about 80 and about 90 bases, e.g., about 85 bases inlength. In some embodiments, these are very short reads, i.e., less thanabout 50 or about 30 bases in length. After obtaining sequence reads,they are further processed as illustrated in FIG. 1.

FIG. 1 is a flow diagram illustrating one embodiment of a method fordetermining copy number state of one of more genomic regions of interestin a sample. The method 100 includes obtaining sequence reads (operation102) and normalizing read counts (operation 104). As described ingreater detail herein, read counts for a genomic region of interest arenormalized with respect to an internal control DNA. The method 100further includes comparing normalized read counts to the internalcontrol DNA (operation 106), thereby obtaining a ratio. The methodfurther includes determining a copy number state of the genomic regionof interest (operation 108) based on the comparison, specifically theratio.

The plurality of reads generated by the sequencing method describedabove are analyzed to determine copy number states, and ultimately copynumber variation, in any of the genomic regions of interest (e.g., SMN1and SMN2) that would necessarily indicate the presence of an autosomalrecessive trait in which copy number variation is diagnostic (e.g.,spinal muscular atrophy). Analysis of the read counts is carried outusing Illumina's HiSeq BclConverter software. Files (e.g., qSeq files)may be generated for both the genomic and barcode reads. In particular,in accordance with one method of the present invention, genomic readdata for each sample is split based upon the barcode reads, which yieldsseparate FASTQ files for each sample.

Analysis of the sequence results has a first step of normalizing theread counts for the SMN1 and SMN2 loci (genetic regions of interest forSMA). The read counts are normalized by dividing the read counts with aread count sum for a control. The read count sum generally includes all17 SMA control loci of the 7 control samples. Then, the averagenormalized values for a set of pre-determined or empirically-identified(e.g., by analysis iteration) wild-type control samples are obtained.Then the normalized read counts for each test sample (each locus) arecompared to the normalized read counts for each of the control samples,thereby obtaining a ratio of normalized read count of testsamples/normalized read count of controls.

Based on the ratios, loci copy numbers may be called as follows: a ratioof <0.1 will be called a copy number state of 0; a ratio between 0.1 and0.8 will be called a copy number state of 1; a ratio between 0.8 and1.25 will be called a copy number state of 2; and a ratio of >1.25 willbe called a copy number state of 3+.

The determined copy numbers can then be used to determine the carrierstatus of an individual from which the sample was obtained (i.e. whetherthe patient is a carrier of the disease). In particular, if the copynumber state is determined to vary from the normal copy state (e.g., CNis 0, 1 or 3+), it is indicative the condition (e.g., carrier of SMA).

Additionally, or alternatively, upon initial normalization of the readcounts for the test samples and control samples, the resulting vector ofnormalized frequencies may include x=[f1, f2, . . . , fn] whichcorrespond to the frequencies of each of the loci being queried (testand control). The normalized frequencies from either a single controlsample or a “synthetic” control (average of multiple control samples)y=[g1, g2, . . . , gn] may be used to calculate the copy number of eachlocus interrogated c=x./y=[f1/g1, f2/g2, . . . , fn/gn].

Functions described above can be implemented using software, hardware,firmware, hardwiring, or combinations of any of these. Any of thesoftware can be physically located at various positions, including beingdistributed such that portions of the functions are implemented atdifferent physical locations.

As one skilled in the art would recognize as necessary or best-suitedfor performance of the methods of the invention, a computer system 200for implementing some or all of the described inventive methods caninclude one or more processors (e.g., a central processing unit (CPU) agraphics processing unit (GPU), or both), main memory and static memory,which communicate with each other via a bus.

In an exemplary embodiment shown in FIG. 2, system 200 includes asequencer 201 with a data acquisition module 205 to obtain sequence readdata. The sequencer 201 may optionally include or be operably coupled toits own, e.g., dedicated, sequencer computer 233 (including aninput/output mechanism 237, one or more of processor 241, and memory245). Additionally or alternatively, the sequencer 201 may be operablycoupled to a server 213 or computer 249 (e.g., laptop, desktop, ortablet) via a network 209. As previously described herein, the sequencer201 may include the HiSeq 2500/1500 system sold by Illumina, Inc. (SanDiego, Calif.).

The computer 249 includes one or more processors 259 and memory 263 aswell as an input/output mechanism 254. Where methods of the inventionemploy a client/server architecture, steps of methods of the inventionmay be performed using the server 213, which includes one or more ofprocessors 221 and memory 229, capable of obtaining data, instructions,etc., or providing results via an interface module 225 or providingresults as a file 217. The server 213 may be engaged over the network209 by the computer 249 or the terminal 267, or the server 213 may bedirectly connected to the terminal 267, which can include one or moreprocessors 275 and memory 279, as well as an input/output mechanism 271.

The system or machines 200 according to the invention may furtherinclude, for any of I/O 249, 237, or 271, a video display unit (e.g., aliquid crystal display (LCD) or a cathode ray tube (CRT)). Computersystems or machines used to implement some or all of the invention canalso include an alphanumeric input device (e.g., a keyboard), a cursorcontrol device (e.g., a mouse), a disk drive unit, a signal generationdevice (e.g., a speaker), a touchscreen, an accelerometer, a microphone,a cellular radio frequency antenna, and a network interface device,which can be, for example, a network interface card (NIC), Wi-Fi card,or cellular modem.

Memory 263, 245, 279, or 229 can include one or more machine-readabledevices on which is stored one or more sets of instructions (e.g.,software) which, when executed by the processor(s) of any one of thedisclosed computers can accomplish some or all of the methodologies orfunctions described herein. The software may also reside, completely orat least partially, within the main memory and/or within the processorduring execution thereof by the computer system.

While the machine-readable devices can in an exemplary embodiment be asingle medium, the term “machine-readable device” should be taken toinclude a single medium or multiple media (e.g., a centralized ordistributed database, and/or associated caches and servers) that storethe one or more sets of instructions and/or data. These terms shall alsobe taken to include any medium or media that are capable of storing,encoding, or holding a set of instructions for execution by the machineand that cause the machine to perform any one or more of themethodologies of the present invention. These terms shall accordingly betaken to include, but not be limited to one or more solid-state memories(e.g., subscriber identity module (SIM) card, secure digital card (SDcard), micro SD card, or solid-state drive (SSD)), optical and magneticmedia, and/or any other tangible storage medium or media.

The invention solves problems associated with current carrier screeningassays by providing molecular inversion probes for capturing at leastone genomic region known or suspected to be associated with a diseaseand subsequently sequencing the captured DNA to determine the copynumber state of the captured DNA. In certain aspects, the captured DNAmay be sequenced by known high-throughput sequence methods ortechniques. The carrier status for the disease can then be determinedbased on the identified copy number state. In particular, the copynumber state may be indicative of whether the patient is a carrier of aparticular disease or condition for which copy number variation isdiagnostic, such as an autosomal recessive trait (e.g., spinal muscularatrophy). For example, it can be determined whether copy numbervariation exists in the genomic regions of interest based on theidentified copy number, thereby providing a method for determiningwhether the patient is a carrier of such an autosomal recessive trait.

Accordingly, the invention overcomes the problems of current carrierscreening assays, particularly MLPA-based assays, based, at least inpart, on compatibility with automated high-throughput screening. Inparticular, the invention provides the sensitivity and specificity fordetection of copy number variation in SMN1 and/or SMN2, similar toMLPA-based methods, by utilizing molecular inversion probes.Additionally, by using high-throughput screening methods, the inventionallows detection of deleterious SMN1 point mutations and indels thatwould otherwise be missed by MLPA and related approaches, therebyproviding more reliable and accurate diagnosis.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

What is claimed is:
 1. A method for determining copy number state of oneor more genes in a sample, the method comprising the steps of: exposinga sample to a plurality of molecular inversion probes capable ofcapturing DNA from at least one genomic region suspected of having analtered copy number and at least one internal control DNA known orsuspected to have a stable copy number; capturing DNA that binds saidmolecular inversion probes; sequencing DNA obtained in said capturestep; normalizing read counts obtained in said sequencing step for saidat least one genomic region with respect to said internal control DNA;comparing said normalized read counts to read counts for at least onecontrol diploid locus; and determining a copy number state of said atleast one genomic region.
 2. The method of claim 1, wherein saiddetermining step comprises determining differences between saidnormalized read counts for said at least one genomic region and saidread counts for said at least one control diploid locus based upon saidcomparison.
 3. The method of claim 1, wherein said normalized readcounts for said at least one control diploid locus are obtained fromsaid sample.
 4. The method of claim 1, wherein said normalized readcounts for said at least one control diploid locus are obtained from aseparate sample that is different from said sample.
 5. The method ofclaim 4, wherein said normalized read counts for said at least onecontrol diploid locus are obtained in silico.
 6. The method of claim 4,wherein said normalized read counts for said at least one controldiploid locus are obtained from a synthetic nucleic acid.
 7. The methodof claim 1, wherein said at least one genomic region comprises a firstgene and a second gene, wherein said first and second genes arehomologs, orthologs, or paralogs.
 8. The method of claim 7, wherein saidfirst gene is SMN1 and said second gene is SMN2.
 9. The method of claim8, further comprising the step of diagnosing a carrier phenotype forspinal muscular atrophy.
 10. The method of claim 1, wherein said readcounts for said at least one control diploid locus are determinedempirically.
 11. The method of claim 1, wherein said sequencing stepcomprises a Sanger sequencing method or a next-generation sequencingmethod.
 12. The method of claim 1, wherein said determining stepcomprises determining a difference between said copy number state ofsaid at least one genomic region to a copy number distributionencompassing a plurality of stable control loci.
 13. The method of claim1, wherein said sample is selected from blood and tissue.
 14. A methodof determining copy number between two paralogous genes, the methodcomprising the steps of: exposing a sample comprising a first geneticlocus, a second genetic locus, and at least one control genetic locus toa plurality of molecular inversion probes, at least some of which arecapable of hybridizing with one or more of said first genetic locus,said second genetic locus, and said control genetic locus; obtaining DNAthat is hybridized to a member of said plurality of molecular inversionprobes; sequencing said DNA; normalizing read counts from saidsequencing step for each of said first genetic locus and said secondgenetic locus with respect to said at least one control genetic locus;comparing normalized read counts for each of said first genetic locusand said second genetic locus to normalized read counts for one or morecontrol samples with a known number of copies of said first geneticlocus and said second genetic locus; and determining relative copynumber of said first genetic locus and said second genetic locus basedupon said comparing step.
 15. The method of claim 14, wherein said firstgenetic locus is SMN1 and said second genetic locus is SMN2.
 16. Themethod of claim 14, wherein said at least one control genetic locus isone that has a stable copy number.
 17. The method of claim 14, furthercomprising the step of genotyping DNA from said sample.
 18. The methodof claim 17, wherein said genotyping step is conducted in the same assayas said copy number determination.
 19. The method of claim 17, whereinsaid genotyping step comprises assessing a genotype at one or more lociknown or suspected to be associated with a disease for which copy numbervariation is diagnostic.
 20. The method of claim 19, wherein saiddisease is spinal muscular atrophy.
 21. The method of claim 19, whereinsaid loci are selected from SMN1, SMN2 and loci different from SMN1 andSMN2.
 22. The method of claim 17, further comprising reporting carrierstatus for one or more conditions.
 23. The method of claim 14, furthercomprising determining carrier status for spinal muscular atrophy. 24.The method of claim 23, further comprising determining carrier statusfor at least one other autosomal recessive trait.
 25. The method ofclaim 24, wherein said at least one other autosomal recessive trait isselected from cystic fibrosis, sickle cell anemia, tay sachs disease,Familial hyperinsulinism, Canavan Disease, Maple Syrup Urine Disease,Bloom's Syndrome, Usher Syndrome type IIIA, Dihydrolipoamidedehydrogenase deficiency, Fanconi anemia group C, Familial dysautonomia,Mucolipidosis Type IV, Usher Syndrome Type IV, Nieman-Pick disease typeA/B, Walker Warburg syndrome, and Joubert Syndrome.
 26. A method fordetermination of carrier status with respect to one or more conditions,the method comprising the steps of: exposing a sample to a plurality ofmolecular inversion probes, at least some of which are capable ofhybridizing to DNA of a first locus or DNA of a second locus; obtainingDNA in said sample that is hybridized to a member of said plurality ofprobes; sequencing said DNA, thereby to obtain sequence read counts forsaid first locus and said second locus; normalizing said read countswith respect to a control locus, the copy number of which is stable;comparing normalized read counts for said first locus and said secondlocus with a standard; and inferring copy number status of said firstlocus and said second locus based upon said comparing step.
 27. Themethod of claim 26, further comprising obtaining a genotype of a thirdlocus.
 28. The method of claim 26, further comprising obtaining agenotype of one or both of said first locus and said second locus. 29.The method of claim 26, wherein said first and second loci areimplicated in an autosomal recessive disorder.
 30. The method of claim29, wherein said first locus is SMN1 and said second locus is SMN2. 31.A method for determination of carrier status with respect to acondition, the method comprising the steps of: exposing a sample to aplurality of molecular inversion probes, at least some of which arecapable of hybridizing to DNA at each of a plurality of loci; obtainingDNA in said sample that is hybridized to a member of said plurality ofprobes; sequencing said DNA, thereby to obtain sequence read counts forat least some members of said plurality of loci; normalizing said readcounts with respect to a control locus, the copy number of which isstable; comparing normalized read counts for members of said pluralitywith a standard; and inferring copy number status of at least one memberof said plurality based upon said comparing step.